Quantitative measurement of various dehydrogenases, such as lactate dehydrogenase (abbreviated occasionally as "LDH" hereinafter in the specification), alcohol dehydrogenase, and glutamate dehydrogenase, have conventionally been conducted by using tetrazolium salt compounds. A property of the tetrazolium salt compound is its ability to receive a hydrogen released by the action of a dehydrogenase, among variety types as mentioned above, via an intermediate electron transporter such as reduced nicotinamide-adenine dinucleotide (abbreviated occasionally as "NADH" hereinafter in the specification) to give a corresponding formazan compound. Accordingly, dehydrogenases can be quantitatively determined by measuring the absorbance of the resulting formazan compound.
Among these dehydrogenases, lactate dehydrogenase distributes all over somatic cells, and in particular, abundantly exists in myocardia, livers, skeletal muscles, and kidneys. It is known that serum LDH activity markedly increases in patients suffered from diseases such as myocardial infarct, malignant tumor, hepatic failure, progressive muscular atrophy, intravascular hemolysis, and megaloblastic anemia. Accordingly, by measuring serum LDH activity, highly useful clinical information for diagnosis can be obtained.
In recent years, in order to detect trace substances in blood, e.g., uric acid and bile acid, with high sensitivity, it has been desired to develop a method for measuring a dehydrogenase which is less susceptible to biogenous substances. For this purpose, 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-ph enyl]-2H-tetrazolium chloride (abbreviated occasionally as "nitro-TB" hereinafter in the specification) and other have generally been used as hydrogen acceptors.
However, the formazan compound formed by nitro-TB after the acceptance of a hydrogen has low water solubility, which causes a practical problem. In particular, in automatic analysis, the resulting formazan compound precipitates inside a measuring system including tubes and cells, and may add positive errors on measured values. In order to solve the problem, it has been desired to develop a method utilizing a tetrazolium salt which produces a water-soluble formazan compound.
As water-soluble tetrazolium salt compounds which form formazan compounds having sufficient solubility, Japanese Patent Unexamined Publication No.(Hei)7-70092/1995 discloses compounds of the following general formula (2): ##STR2## wherein R.sup.1 and R.sup.2 independently represent hydrogen atom or nitro group, and M represents an alkali metal or an ammonium.
Since these compounds produce formazan compounds having extremely high detection sensitivity, measurement with higher sensitivity can be achieved by using these compound compared to the conventionally used nitro-TB. In addition, because of the water-solubility of the formazan compounds, they are free from adhesion to a measuring apparatus, and accordingly, useful for clinical diagnostics. In particular, the compounds of the above general formula wherein R.sup.1 and R.sup.2 are nitro groups have a feature of efficient reactivity with NADH to give a formazan compound exhibiting extremely high absorbance.
However, researches by the inventors of the present invention revealed that these compounds have a problem of low storage stability when stored in the state of an aqueous solution. Because aqueous solutions are often stored, after their preparation, for a long period of time such as for 3 to 6 months in usual clinical analyses, it is desired that tetrazolium compounds used as hydrogen acceptors have high storage stability in the state of an aqueous solution.